Total Protein Detection

Overview

Total protein staining provides an image of the protein migration pattern in a gel or on a blot. This information helps determine transfer efficiency and the molecular weight, relative quantity, and other properties of the transferred proteins. This section provides an overview of total protein stains, including anionic dyes (such as amido black, Coomassie blue, Ponceau S, and Fast Green), fluorescent stains, and colloidal gold stains and discusses the advantages and disadvantages of these stains. It also provides protocols for total protein staining and troubleshooting tips for various problems in staining procedures.

Considerations for Protein Detection

Protein standards are useful for monitoring transfer efficiency and serve as molecular weight markers for calibration of blot patterns. Refer to protein standards section for available Bio-Rad protein standards.

Polyacrylamide gels shrink during staining, so comparison of an immunologically probed membrane to a stained gel is not practical. To determine the exact location of a specific antigen in relation to other proteins, compare two blotted membranes, one that has been probed with an antibody and the other stained for total protein.

Total protein detection. Blot stained with SYPRO Ruby blot stain showing the total protein pattern of an E.coli lysate containing an over-expressed GST fusion protein on the blot.

Anionic Dyes

The first techniques developed for total protein staining of blotted membranes used the same anionic dyes commonly used for staining proteins in polyacrylamide gels. These dyes include amido black (Towbin et al. 1979), Coomassie (Brilliant) Blue R-250 (Burnette 1981), Ponceau S, and Fast Green FCF (Reinheart and Malamud 1982). Of these:

Fluorescent Stains

Fluorescent stains such as SYPRO Ruby and Deep Purple provide highly sensitive detection of proteins on blots as well as in gels. SYPRO Ruby blot stain allows detection as low as 2 ng. After staining, target proteins can be detected by colorimetric or chemiluminescence immunodetection methods, or analyzed by microsequencing or mass spectrometry with no interference from the protein stain.

Colloidal Gold Stain

Colloidal gold is an alternative to anionic dyes that provides detection sensitivities rivaling those of immunological detection methods (Moeremans et al. 1987, Rohringer and Holden 1985). When a solution of colloidal gold particles is incubated with proteins bound to a nitrocellulose or PVDF membrane, the gold binds to the proteins through electrostatic adsorption. The resulting gold-protein complex produces a transient, reddish-pink color due to the optical properties of colloidal gold. This gold-protein interaction is the basis for total protein staining with colloidal gold as well as for specific, immunogold detection (see Immunogold Labeling)

Replace or thoroughly clean contaminated fiber pads if a tank blotter was used

Excessive amounts of protein were loaded on the gel or too much SDS was used in the transfer buffer. Proteins can pass through the membrane without binding and recirculate through a tank blotting system

Reduce the amount of protein on the gel or SDS in the transfer buffer

Add a second sheet of membrane to bind excess protein

The colloidal gold stain solution was contaminated

Use a separate, clean plastic container to store previously used reagent in the refrigerator

Discard any reagent that has a viscous sediment at the bottom of the bottle

If the solution is no longer dark burgundy but light blue, discard it. The stain is contaminated with buffer salts, which react with the gold solution, causing nonspecific precipitation of the reagent onto the membrane

The development step was too long

Overnight development may slightly increase sensitivity but may also increase background. Reduce development step to 1–2 hr

Colloidal gold total protein stain — low sensitivity

The incubation time was insufficient

Increase the incubation time for detection of low-level signals. Overnight incubation is possible, although background staining can increase

Increase the number and duration of washes with the destaining solution

The dye solution was too concentrated

Prepare new solution

Anionic dyes — low sensitivity

Anionic dye stains do not detect protein bands below ~100 ng

Use a more sensitive stain such as colloidal gold stain or a fluorescent stain

Increase the sample load

Fluorescent blot stains — low sensitivity

Proteins with low hydrophobicity

Only highly hydrophobic proteins will retain enough SYPRO stain to be visible on a membrane. SDS is stripped off proteins during transfer, resulting in very little retention of the SYPRO stain on most proteins

Incorrect excitation and emission settings were used

Refer to the product literature for correct excitation wavelengths and emission filters

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